Benign prostatic hyperplasia (BPH) and enlargement is a highly prevalent condition amongst elderly men and is frequently associated with lower urinary tract symptoms (LUTS).  The aetiology of BPH-LUTS is multifactorial and is believed to result from concurrent physiological and metabolic alterations that arise with age.  Cytochrome B5 reductase type-3 (CYB5R3) is an oxidoreductase that regulates pathways in the mitochondria, endoplasmic reticulum and nitric oxide-cyclic GMP signalling through its actions on soluble guanylate cyclase. Downregulation of CYB5R3 activity has been associated with oxidative stress, inflammation, and metabolic dysfunction [1], all of which progressively increase through natural aging. We hypothesized that selective knockdown of CYB5R3 in the prostate would induce metabolic dysfunction comparable to advance age resulting in a BPH-LUTS phenotype.  To address this hypothesis, we generated a conditional CYB5R3 knockout mouse and performed 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) injections into the prostate lobes. The voiding activity of mice was monitored over 12 weeks and prostate after which tissues were examined for morphological changes by histology.  The purpose of this study was to generate an animal model of BPH-LUTS by mimicking the cellular dysfunctions associated with aging selectively within the prostate.

Generation of CYB5R3 conditional knockout mouse. A mouse with loxP sites flanking exon3 of the CYB5R3 gene [2] (CYB5R3flox/flox) was crossed with a mouse expressing the tamoxifen inducible Cre recombinase under the β-actin promoter (CAG-Cre, Jackson laboratories, stock#:004682) to generate the conditional CYB5R3flox/flox+CAG-Cre (CYB5R3 KO) mouse (Fig 1A).  Both mouse strains were based on a C57Bl/6 background.  At 8-12 weeks of age, male CYB5R3 KO and age matched CYB5R3 wildtype (WT) mice were used for 4-OHT prostate injections.  Mice were anesthetized with isoflurane and using sterile surgical conditions a lower midline incision was made to expose the prostate glands and urinary bladder.  Each lobe was bilaterally injected with 2-5 µl of 0.5 mg/ml 4-OHT (dissolved in ethanol/CremophorEL/saline) using a 32-gauge insulin syringe (Fig 1B).  The incisions were sutured, and mice were given prophylactic ampicillin (100 mg/kg, SQ, 7 days) and ketoprofen (3 mg/kg, IM, 3 days) during the recovery period.

Voiding assessments.  Voiding activity was initially analyzed by two-hour urine spot tests starting one week prior to 4-OHT injections and performed weekly thereafter for 6 weeks.  At 8 weeks after surgery, weekly 24-hour metabolic cage assessments were performed for 13 weeks after injections. Metabolic cages (Columbus Instruments Inc.) were maintained in a climate-controlled cabinet on 12-hour light/dark cycles. 

Histology and immunofluorescence.  Following completion of voiding assessments, mice were humanely sacrificed for tissue collection.  Prostate tissues were fixed in 10% neutral buffered formalin and processed for paraffin embedding, sectioned 3-4 µm thick and processed for Van Gieson staining and immunofluorescence.  Slides were imaged using an Olympus BX63 microscope and analysis performed using FIJI ImageJ software.

Data and statistical analysis. Data are expressed as mean ± standard deviation.  Pairwise comparisons were performed using Student’s t-test where the null hypothesis was rejected at p<0.05.

Prostate CYB5R3 KO mice exhibit urinary frequency and reduced voided volumes.  CYB5R3 KO and WT mice (N=4 each) began showing differences in voiding behaviour at 10-12 weeks following 4-OHT injections with KO mice exhibiting increased voiding frequency (Fig 1C) and decreased voided volumes (Fig 1D).  There were no differences in total voided volumes (Fig 1E) or total water intake (Fig 1F) between KO and WT mice.  Additionally, there were no significant differences in body weight (Fig 2A) over the entire observation period nor changes in blood pressure monitored by weekly tail cuff plethysmography (not shown), indicating there were little to no systemic effects of 4-OHT injections.

CYB5R3 KO have increased prostate collagen deposition and glandular growth.  Gross examination of the tissues from CYB5R3 KO and WT mice showed no significant differences in bladder or seminal vesical wet weights (Fig 2A).  Van Gieson staining (Fig 2B-2D) showed increased collagen deposition (pink) within the convergence point of the urethra, prostate glands, seminal vesicles, and ductus deferens, all of which are surrounded by skeletal muscle of the external urethral sphincter (EUS).  Moreover, there was increased number of glandular structures further congesting the area (Fig 2D).  Immunofluorescence of prostate sections from KO mice when compared to those of WT (Fig 2F versus 2E) exhibited decreased CYB5R3 (red) localization to the prostate glandular epithelium of the lateral lobes surrounding the EUS (outlined with a dotted white line) and increased α-smooth muscle actin (green) around glands and in foci within the interstitial space (Fig 2F, white arrows).

CYB5R3 knockdown in the prostate resulted in increased urinary frequency and voided volumes which correlated with increased fibrosis and glandular growth within the prostate, without signs of outlet obstruction.  These observations closely match with voiding features seen with aged male mice [3] suggesting disruption of local cellular metabolic processes are a major contributor of BPH-LUTS pathophysiology that precedes outlet obstruction.

Understanding of the mechanisms that drive development of BPH-LUTS are still lacking and use of aged rodent models can be cost prohibitive and highly variable.  Selective CYB5R3 KO may represent a useful animal model to study the milieu of the aging prostate.

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Durgin et al., Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.  JCI Insight, 4(19):e129183, 2019.
Zabbarova et al., Benign prostatic hyperplasia/obstruction ameliorated using a soluble guanylate cyclase activator. J Pathol., 256(4):442-454, 2022.

Continence 2S2 (2022) 100220
DOI: 10.1016/j.cont.2022.100220